Posted by
Olivier Burri on
Jan 17, 2014; 1:19pm
URL: http://imagej.273.s1.nabble.com/BrdU-cell-counting-tp5006086p5006152.html
Dear Josue,
> How many cells do you recommend measuring for a good estimation of
> number of cells? Would a 100 be ok?
As a rule of thumb, 100 cells could yield a good estimate. But this is very dependent on the kind of change you are expecting.
If between conditions your cells change in size but not in number, the approach I suggested is not ideal, as you'll think there are more cells when they actually just got bigger.
This is why a lot of people are satisfied with an area coverage for example, rather than actual cell numbers.
But if you have about 100 cells that are easily measurable per condition, then you can use that average to estimate the number inside the uncountable clusters. Just check that they don't change size between conditions.
> If I estimate the area covered by cells, Will I need to set the scale to a given
> known value? I have a 1 mm rule in the magnification of the photos.
> To measure the cells, Will I also need to have the scale set?
You can convert the pixel values to microns if you want to have the output directly scaled. Or you can do this all the way at the end.
Have all your measurements in pixels, when you estimate the total number of cells, it makes no difference as you're dividing an area in either pixels or microns with the average area of the cell you have either in pixels or microns. This is true as long as you do not have images at different magnifications.
To save you the hassle of putting everything to scale on each image, I'd suggest to do a cross-product at the end.
Otherwise:
If you have a 1mm rule, then you can draw a line profile on the rule and by using: "Analyze --> Set Scale...", you can easily calibrate your image.
As a final note, by the description of your images, there are a couple more questions I would have:
- What format are they? As they are RGB images, you'll need to use 'Color Deconvolution' to extract the DAB channel, but the results are less than optimal if you have a JPEG image. Make sure it's TIFF or some lossless format.
- You mention having a ruler on your image, so you should crop this out before you try to set a threshold, especially if you go for automated methods.
The easiest thing would be for you to send me or point me to one of your images so that we can check if the quality is good for starters.
All the best
Oli
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